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Design, Synthesis and in vitro Biological Evaluation of Acrylamide Derivatives Against Chikungunya Virus

Edeildo F. Silva-Júnior 1,*, Gabriel F.S. Passos 1, Matheus G.M. Gomes 1, Thiago M. Aquino 1, Stephannie J.M. Souza 2, João P.M. Cavalcante

2, Elane C. Santos 2,

Ênio J. Bassi 2, and João X. Araújo-Júnior 1

1 Laboratory of Medicinal Chemistry, Institute of Pharmaceutical Sciences, Federal

University of Alagoas, Maceió, Brazil;

2 Laboratory of Research in Virology and Immunology, Institute of Biological Sciences

and Health, Federal University of Alagoas, Maceió, Brazil.

* Corresponding author: edeildo.junior@esenfar.ufal.br

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Graphical Abstract

Design, Synthesis and in vitro Biological Evaluation of Acrylamide Derivatives Against Chikungunya Virus

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Abstract: Chikungunya virus (CHIKV) causes an infectious disease characterized by inflammation and pain of the musculoskeletal tissues accompanied by swelling in the joints and cartilage damage. Currently, there are no licensed vaccines or chemotherapeutic agents to prevent or treat CHIKV infections. In sense, this research aims to explore the potential in vitro anti-CHIKV activity of acrylamide

• derivatives. In silico techniques were applied to 132 acrylamides toward the six

most important biological targets from CHIKV. Subsequently, ten most promising acrylamides were selected and synthesized. From cytotoxicity MTT assay was verified that GP03, 07, and 09 demonstrate cell viability higher than 94%. Additionally, GP03 and 09 exhibited weak viral inhibition values (50 and 32% at 40 µM, respectively). In contrast, GP07 displayed a significant in vitro anti-CHIKV activity, with inhibition of 81%. Thus, docking simulations were performed to suggest a potential CHIKV-target for GP07. It was observed that the GP07 has a high affinity towards E protein. Moreover, GP07 reduced the percentage of CHIKV- positive cells from 74.07 to 0.88%, 48h post-treatment on flow cytometry. In conclusion, all virtual simulations corroborated with experimental results, and GP07 could be used as a promising anti-CHIKV scaffold for designing new drugs in the future. Keywords: Virtual screening; acrylamides; Chikungunya; antiviral; molecular docking; E protein.

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Introduction – Chikungunya virus key facts

Chikungunya virus (CHIKV) is an Alphavirus transmitted to humans by infected Aedes aegypti and Ae. albopictus mosquitoes. It causes fever, severe joint pain, and cartilage damage.¹  CHIKV shares some clinical signs with Dengue and Zika, and can be misdiagnosed;²,³  Joint pain is often debilitating and can vary in duration;³  There are no licensed vaccines or chemotherapeutic agents to treat or prevent this infectious disease;¹,³  CHIKV has been identified in over 60 countries in Europe, Asia, and the Americas.³

¹ Silva-Júnior et al. 2017. Bioorg & Med Chem, 25(16), 4219-4244. ² Silva-Júnior et al. Bioorg & Med Chem, 27(18), 3963-3978. ³ WHO. 2014. Protect yourself from vector-brone diseases. Silva-Júnior et al. 2017. Bioorg & Med Chem, 25(16), 4219-4244. (adap.)

Introduction – Global distribution for Chikungunya virus

https://www.sciencenews.org/article/chikungunya-move

Introduction – Key macromolecular targets from CHIKV

Immature E Protein E Protein Capsid nsP2 nsP2/helicase nsP3

CHIKV Structural Proteins CHIKV Non- Structural Proteins

These proteins are related to viral structure, adsorption and host cell entry. These proteins are associated with the virus replication.

Introduction – Anti-CHIKV scaffolds found in the literature

* Acylhydrazones * Acrylamides * Acylhydrazines

Tardugno et al., 2018. Bioorg & Med Chem, 26(4), 869-874. Giancotti et al., 2018. Eur J Med Chem, 149, 56-68.

Results and discussion – Rational design for acrylamides

* Molecular targets: nsP2, nsP2/helicase, nsP3, immature E protein, E protein, and capsid.

Acrylamide - The most promising scaffold

Results and discussion – Virtual screening for acrylamides

* Hydrazone compounds (1 and 3) were considered into virtual screening steps to identify chemical characteristics from these molecular class, such as interactions, fitscore values, among others. Molecular targets: nsP2 (PDB: 3TRK), nsP2/helicase (PDB: 6JIM), nsP3 (PDB: 3GPO), immature E protein (PDB: 3N40), E protein (PDB: 3N41), and capsid (PDB: 5H23). In sense, dynamics simulations were performed by using Gromacs (10 ns) and molecular dockings using Gold software (ChemPLP genetic agorithm).

132 possible compounds

(33 R-substituents x 4 scaffolds)

Dynamics

(Gromacs)

Docking

(Gold)

6 molecular targets from CHIKV

Molecular modeling

The 10 most promising R- substituents 2 1 3 4

Results and discussion – Synthesis of new antiviral acrylamides

Initially, cinnamic acids were synthesized by Knoevenagel/Doebner modification reaction using malonic acid (1 eq) and the corresponding aldehydes (1 eq). Cinnamic acids were purified by filtration and washing with concentrated HCl (37%), and collected powders were dryied under high-vaccum. Subsequently, the final compounds (GP’s) were obtained by TBTU-coupling reaction between aniline (1 eq) and the corresponding cinnamic acids (1.1 eq), in DMF at room temperature (overnight), and DIPEA as catalyst base. All purifications were performed by filtration and washing with a satured NaHCO3 solution and destilled water, respectively. In some cases, it was necessary to recrystallize the product from an acetone/water (1:2) mixture.

Cinnamic acids Acrylamides

Results and discussion – Chemical characterization

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*The table shows values for all compounds. Physico-chemical parameter Values (range) Molecular Mass 241.09 – 299.13 g/mol State Solids Retention Time (RT) 2.9 – 3.88 Purity 96.3 – 99.9% Melting Point (Mp) 123 – 246 °C Degradation Point (Dp) > 300 °C

GP compounds*

GP-07 ¹H NMR spectrum (DMSO-d6) 600 MHz

H Ha Hb

Zoom into the acryl peaks region

Ha Hb

6.89 ppm 7.58 ppm 10.23 ppm

Trans (E) configuration acryl group

Results and discussion – Cell viability (MTT assay)

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C o m p o u n d (2 0  M ) % C e ll v ia b ility

G P

• 1

G P

• 2

G P

• 3

G P

• 4

G P

• 5

G P

• 6

G P

• 7

G P

• 8

G P

• 9

G P

• 1

C C 5 0 1 0 0 1 5 0

**** **

The cytotoxicity was performed in vitro for ten acrylamides (GP01-10) toward Vero E6 cells at 20 µM concentration by MTT assay

10 acrylamides GP-01 to GP-10 Vero E6 cells 48h CELL VIABILITY (MTT assay)

GP-02 acrylamides was cytotoxic (≤ 50%)

** p ≤ 0.01; *** ≤ 0.001 vs cell control (CC)

C o m p o u n d (2 0  M )

G P -0 1 G P -0 3 G P -0 4 G P -0 5 G P -0 6 G P -0 7 G P -0 8 G P -0 9 G P -1 0 C H IK V C C 5 0 1 0 0 1 5 0

% In h ib itio n

**** *** **** ** ****

Results and discussion – In vitro antiviral screening

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Anti-CHIKV activity was detected for GP-01, GP-03, GP-07, GP-09 and GP-10 acrylamides at 20 µM

Chikungunya virus (CHIKV)

Vero E6 cells

CHIKV adsorption

Non-cytotoxic acrylamides

48h CELL VIABILITY (MTT assay)

Evaluation of anti-CHIKV activity of acrylamides

Results and discussion

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C o m p o u n d (4 0  M ) % C e ll v ia b ility

G P -0 1 G P -0 3 G P -0 7 G P -0 9 G P -1 0 C C 5 0 1 0 0 1 5 0

**** ****



G P -0 3 G P -0 7 G P -0 9 C H IK V C C 5 0 1 0 0 1 5 0

% In h ib itio n

**** **** ****

The cytotoxicity was performed for five selected acrylamides at 40 µM concentration for 72h As result, it was observed that acrylamides GP03 and 09 exhibited weak viral inhibition values (49 and 32%, respectively). In contrast, the acrylamide GP07 displayed a significant in vitro anti-CHIKV activity, with an inhibition value of 81% after 72h.  GP-1 and GP-10 were cytotoxic after 72h

Evaluation of anti-CHIKV activity of selected acrylamides at 40 µM

Results and discussion – Detection of CHIKV-infected cells

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Chikungunya virus (CHIKV)

Vero E6 cells

CHIKV adsorption 48h

In order to confirm the antiviral activity, the intracellular labelling of CHIKV was performed and the percentage of CHIKV-positive cells was detected by flow cytometry

1) Monoclonal anti-CHIKV antibody 2) Alexa Fluor 488 Anti-mouse IgG Cell fixation and permeabilization followed by staining

Negative control CHIKV

Percentage of CHIKV-positive cells was detected by flow cytometry

GP-07

Results and discussion

16 GP-07 inhibited the CHIKV infection in vitro. (A) Representative micrographs (200x magnification) showing the cytopathic effect and (B) flow cytometry dot-plots of Vero E6 cells infected (CHIKV) or uninfected (Ctrl/negative control) with CHIKV. The cells were treated with GP-07 at 20 and 40 µM. Percentages of CHIKV-positive cells are shown. C) Mean ± SEM of CHIKV-positive cells (triplicate).

% c e lls in fe c te d

C trl C H IK V G P 0 7 2 0 u M G P 0 7 4 0 u M 20 40 60 80

**** **** **** ***

C) As result, GP07 was able to reduce the percentage of CHIKV-positive cells from 74.07 to 0.88 %, 48h post-treatment.

CHIKV-positive cells (%)

Results and discussion – Structure-Activity Relationship (SAR)

 SAR at 40 µM concentration

Best analog (GP-07)

Results and discussion – Molecular target proposal for GP-07

After all molecular docking simulations, E protein was found to be the main target for GP-07. Furthermore, it was confirmed in our experimental results, in which was observed a protective effect on host cell towards CHIKV, suggesting that GP-07 binds to the CHIKV viral surface and prevents its adhesion/adsorption to the cells.

GP-07

In general, it was not verified H-bonds between GP-07 and the binding site from the E protein, suggesting an essential role of hydrophobic groups in the complex formation. Mature E protein glycoprotein complex (E1-E2-E3 ). PDB id: 3N41

Conclusions – Remarkable topics

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 Theoretical data were corrobarated by experimental results;  GP-03 could be also considered as promising compound. However, it needs specific modifications to improve its potential anti-CHIKV activity;  Based on GP-07 results, it is possible to suggest that this compound could be used as a promising anti-CHIKV scaffold for designing new antiviral agents in the future.

Compound Selection and Synthesis

• From 132

compounds, we have identified 10 promising compounds (acrylamides) to be synthesized Cytotoxicity Assay

• 9 of 10

compounds presented good tolerance at 20 µM concentration Viral Inhibition Assay

• GP-07 was

found to be the most active analog towards infected cell with CHIKV in vitro Target Proposal

• Docking

simulations were able to predicted that GP-07 binds to the CHIKV E protein.

Additional points & perspectives

Acknowledgments

The authors thank the National Research Council (CNPq protocol number: 437407/2018-7), Foundation for Innovation and Research (FINEP), Foundation of Support to Research from the State of Alagoas (FAPEAL), and CAPES Foundation (protocol number: 88887.355610/2019-00) for their financial support. The authors also acknowledge the website https://smart.servier.com used to generate some charts.

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